Bovine b-endothelial cell growth factor

ABSTRACT

Bovine β-endothelial cell growth factor (β-ECGF) having an apparent molecular weight of 20,000 daltons can be purified at least 16,300 fold from bovine brain using heparin-Sepharose affinity chromatography. ECGF is useful for, among other purposes, diagnostic applications and has potential in the treatment of damaged blood vessels or other endothelial cell-lined structures.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application Ser. No. 07/799,859 now abandoned,filed on Nov. 27, 1991; which is a continuation of application Ser. No.07/693,079, filed on Apr. 29, 1991 now abandoned which is a continuationof Ser. No. 07/134,499 filed Dec. 18, 1987 and now abandoned, which is acontinuation-in-part of application Ser. No. 835,594, filed Mar. 3,1986, now U.S. Pat. No. 4,868,113.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to naturally-derived bovine endothelial cellgrowth factor (ECGF). More particularly, this invention relates to thebovine β-ECGF and the use of ECGF in the treatment of endothelial celldamage and/or regeneration.

2. The Prior Art

Endothelial cell growth factor, referred to herein as "ECGF" is amitogen for endothelial cells in vitro. Growth of endothelial cells is anecessary step during the process of angiogenesis [Maciag, Prog.Hemostasis and Thromb., 7:167-182 (1984); Maciag, T., Hoover, G. A., andWeinstein, R., J. Biol. Chem., 257: 5333-5336 (1982)]. Bovine ECGF hasbeen isolated by Maciag, et al., [Science 225:932-935 (1984)] usingstreptomycin sulfate precipitation, gel. exclusion chromatography,ammonium sulfate precipitation and heparin-Sepharose affinitychromatography. Bovine ECGF purified in this manner yields asingle-chain polypeptide which possesses an anionic isoelectric pointand an apparent molecular weight of 20,000 [Maciag, supra; Schreiber, etal., J. Cell Biol., 101:1623-1626 (1985); and Schreiber, et al., Proc.Natl. Acad. Sci. USA, 82:6138-6142 (1985)]. Recently, murine monoclonalantibodies against bovine ECGF have been produced (Maciag, et al.,supra) which may be useful in purifying bovine ECGF in a manner similarto the monoclonal antibody purification of Factor VIIIC described byZimmerman and Fulcher in U.S. Pat. No. 4,361,509.

In general, recombinant DNA techniques are known. See Methods InEnzymology, (Academic Press, New York) volumes 65 and 68 (1979); 100 and101 (1983) and the references cited therein, all of which areincorporated herein by reference. An extensive technical discussionembodying most commonly used recombinant DNA methodologies can be foundin Maniatis, et al., Molecular Cloning, Cold Spring Harbor Laboratory(1982). Genes coding for various polypeptides may be cloned byincorporating a DNA fragment coding for the polypeptide in a recombinantDNA vehicle, e.g., bacterial or viral vectors, and transforming asuitable host. This host is typically an Escherichia coli (E. coli)strain, however, depending upon the desired product, eukaryotic hostsmay be utilized. Clones incorporating the recombinant vectors areisolated and may be grown and used to produce the desired polypeptide ona large scale.

Several groups of workers have isolated mixtures of messenger RNA (mRNA)from eukaryotic cells and employed a series of enzymatic reactions tosynthesize double-stranded DNA copies which are complementary to thismRNA mixture. In the first reaction, mRNA is transcribed into asingle-stranded complementary DNA (ss-c DNA) by an RNA-directed DNApolymerase, also called reverse transcriptase. Reverse transcriptasesynthesizes DNA in the 5'-3' direction, utilizes deoxyribonucleoside5'-triphosphates as precursors, and requires both a template and aprimer strand, the latter of which must have a free 3'-hydroxylterminus. Reverse transcriptase products, whether partial or completecopies of the mRNA template, often possess short, partiallydouble-stranded hairpins ("loops") at their 3' termini. In the secondreaction, these "hairpin loops" can be exploited as primers for DNApolymerases. Preformed DNA is required both as a template and as aprimer in the action of DNA polymerase. The DNA polymerase requires thepresence of a DNA strand having a free 3'-hydroxyl group, to which newnucleotides are added to extend the chain in the 5'-3' direction. Theproducts of such sequential reverse transcriptase and DNA polymerasereactions still possess a loop at one end. The apex of the loop or"fold-point" of the double-stranded DNA, which has thus been created, issubstantially a single-strand segment. In the third reaction, thissingle-strand segment is cleaved with the single-strand specificnuclease S1 to generate a "blunt-end" duplex DNA segment. This generalmethod is applicable to any mRNA mixture, and is described by Buell, etal., J. Biol. Chem., 253:2483 (1978).

The resulting double-stranded cDNA mixture (ds-cDNA) is inserted intocloning vehicles by any one of many known techniques, depending at leastin part on the particular vehicle used. Various insertion methods arediscussed in considerable detail in Methods In Enzymology, 68:16-18(1980), and the references cited therein.

Once the DNA segments are inserted, the cloning vehicle is used totransform a suitable host. These cloning vehicles usually impart anantibiotic resistance trait to the host. Such hosts are generallyprokaryotic cells. At this point, only a few of the transformed ortransfected hosts contain the desired cDNA. The sum of all transformedor transfected hosts constitutes a gene "library". The overall ds-cDNAlibrary created by this method provides a representative sample of thecoding information present in the mRNA mixture used as the startingmaterial.

If an appropriate oligonucleotide sequence is available, it can be usedto identify clones of interest in the following manner. Individualtransformed or transfected cells are grown as colonies on anitrocellulose filter paper. These colonies are lysed; the DNA releasedis bound tightly to the filter paper by heating. The filter paper isthen incubated with a labeled oligonucleotide probe which iscomplementary to the structural gene of interest. The probe hybridizeswith the cDNA for which it is complementary, and is identified byautoradiography. The corresponding clones are characterized in order toidentify one or a combination of clones which contain all of thestructural information for the desired protein. The nucleic acidsequence coding for the protein of interest is isolated and reinsertedinto an expression vector. The expression vector brings the cloned geneunder the regulatory control of specific prokaryotic or eukaryoticcontrol elements which allow the efficient expression (transcription andtranslation) of the ds-cDNA. Thus, this general technique is onlyapplicable to those proteins for which at least a portion of their aminoacid or DNA sequence is known for which an oligonucleotide probe isavailable. See, generally, Maniatis, et al., supra.

More recently, methods have been developed to identify specific clonesby probing bacterial colonies or phage plaques with antibodies specificfor the encoded protein of interest. This method can only be used with"expression vector" cloning vehicles since elaboration of the proteinproduct is required. The structural gene is inserted into the vectoradjacent to regulatory gene sequences that control expression of theprotein. The cells are lysed, either by chemical methods or by afunction supplied by the host cell or vector, and the protein isdetected by a specific antibody and a detection system such as enzymeimmunoassay. An example of this is the lambda gt 11 system described byYoung and Davis, Proc. Nat'l. Acad. Sci. USA, 80:1194-1198 (1983) andYoung and Davis, Science, 222:778 (1983).

SUMMARY OF THE INVENTION

Multiple forms of bovine ECGF have been isolated [Burgess, et al., J.Biol. Chem. 260:11389-11392 (1985)] by sodium chloride gradient elutionof bovine ECGF from the heparin-Sepharose column or by reversed-phasehigh pressure liquid chromatography (HPLC). The two isolatedpolypeptides, designated as alpha- and beta-ECGF, have apparentmolecular weights of 17,000 and 20,000, respectively. Using thisprocedure, the bovine ECGF contained in 8,500 ml of bovine brain extract(6.25×10⁷ total units) is concentrated into a total of 6 ml ofalpha-ECGF (3.0×10⁶ units) and 3 ml of beta-ECGF (5.2×10⁵ units). Thisis a 9,300-fold purification of alpha-ECGF and 16,300 fold purificationof beta-ECGF (Burgess supra).

The present invention has also made it possible to provide readilyavailable, large quantities of ECGF or ECGF fragments. This has beenachieved with oligonucleotides whose design was based upon knowledge ofthe amino acid sequence of bovine ECGF and which react specifically withthe ECGF cDNA. Production of ECGF is achieved through the application ofrecombinant DNA technology to prepare cloning vehicles encoding the ECGFprotein and procedures for recovering ECGF protein essentially free ofother proteins of human origin.

Accordingly, the present invention provides ECGF or its fragmentsessentially free of other proteins of human origin. ECGF is produced byrecombinant DNA techniques in host cells or other self-replicatingsystems and is provided in essentially pure form. The invention furtherprovides replicable expression vectors incorporating a DNA sequenceencoding ECGF and a self-replicating host cell system transformed ortransfected thereby. The host system is usually of prokaryotic, e.g., E.coli or B. subtilis, or eukaryotic cells.

The ECGF is produced by a process which comprises (a) preparing areplicable expression vector capable of expressing the DNA sequenceencoding ECGF in a suitable host cell system; (b) transforming said hostsystem to obtain a recombinant host system; (c) maintaining saidrecombinant host system under conditions permitting expression of saidECGF-encoding DNA sequence to produce ECGF protein; and (d) recoveringsaid ECGF protein. Preferably, the ECGF-encoding replicable expressionvector is made by preparing a ds-cDNA preparation representative of ECGFmRNA and incorporating the ds-cDNA into replicable expression vectors.The preferred mode of recovering ECGF comprises reacting the proteinsexpressed by the recombinant host system with a reagent compositioncomprising at least one binding site specific for ECGF. ECGF may be usedas a therapeutic agent in the treatment of damaged or in regeneratingblood vessels and other endothelial cell-lined structures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a general procedure for enzymatic reactions toproduce cDNA clones.

FIG. 2 illustrates the production of a library containing DNA fragmentsinserted into lambda gt₁₁.

FIG. 3 illustrates a partial amino acid sequence of bovine alpha andbeta ECGF.

Line a: Amino-terminal amino acid sequence of bovine alpha ECGF.

Line b: Amino-terminal amino acid sequence of bovine beta ECGF. Thesequence beginning with PheAsnLeu . . . was determined fromtrypsin-cleaved bovine beta ECGF.

Line c: Amino acid sequence of cyanogen bromide-cleaved bovine alphaECGF.

Line d: Amino acid sequence of cyanogen bromide-cleaved bovine betaECGF.

FIG. 4 illustrates hydrogen-bonded base pairs.

FIG. 5 illustrates the design of an oligonucleotide probe for humanEndothelial Cell Growth Factor.

FIG. 6 illustrates a schematic diagram of human ECGF cDNA clones 1 and29. The open reading box represents the open reading frame encodinghuman beta ECGF. The EcoRI sites correspond to synthetic oligonucleotidelinkers used in the construction of the cDNA library. The poly (A) tailat the 3' end of clone 1 is shown by A17.

FIG. 7 illustrates homology between human ECGF cDNA sequence andoligonucleotide probes.

Line a: Bovine trypsin- or cyanogen bromide-cleaved beta ECGF amino acidsequence.

Line b: Unique oligonucleotide probe.

Line c: Human ECGF cDNA sequence (determined from lambda ECGF clones 1and 29).

Line d: Human ECGF amino acid sequence, deduced from cDNA sequenceanalysis.

FIG. 8 illustrates the complete cDNA sequence of human ECGF. The cDNAinserts from ECGF clones 1 and 29 were subcloned into M13mp18 and theECGF-encoding open reading frame and flanking regions sequenced by thechain termination method. In frame stop codons at the 5' and 3' ends ofthe ECGF-encoding open reading frame are indicated by the underlinedsequence and trm, respectively. The single-letter notation for aminoacids is used: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His;I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser;T, Thr; V, Val; W, Trp; Y, Tyr.

FIG. 9 illustrates Northern blot analysis of ECGF mRNA. RNA wasdenatured in 2.2M formaldehyde and 50% formamide and fractionated byelectrophoresis in a 1.25% agarose gel containing 2.2M formaldehyde.This was transferred to GENESCREEN PLUS (New England Nuclear) byblotting with 1OX SSPE. Blots were hybridized to ³² P-labelednick-translated probes of ECGF clone 1 at 65° C. for 16 hours in amixture containing 2× SSPE, 20× Denhardt's solution, yeast transfer RNA(200 μg/ml), and 0.2% SDS. The membrane was subsequently washed at 65°C., twice with 2× SSPE and 0.2% SDS, then twice with 0.2× SSPE and 0.2%SDS, air-dried, and exposed overnight to Kodak XAR film with anintensifying screen. The migration of 28S and 18S RNA is noted.

Lane 1: 10 μg human brain poly(A)-containing RNA.

Lane 2: 10 μg human adult liver poly(A)-containing RNA.

FIG. 10 illustrates expressional cloning of human recombinant α-ECGF.The expression vector pMJ26 was constructed as indicated. Thetranslation initiation codon provided by the synthetic oligonucleotideis indicated by "ATG". The hybrid tac promoter and the Shine-Dalgarnosequence provided by the vector pKK223-3, are indicated by "Ptac" andS.D.", respectively Transcription terminators are indicated by "rrnBT₁T₂ " and "5S". The open arrow shows the direction of transcription fromthe tac promoter.

FIG. 11 illustrates SDS-PAGE analysis of recombinant human α-ECGFexpression and purification. Cultures of pMJ26 in E. coli JM103 weregrown and induced with 1 mM IPTG. Lanes a and b, samples lysed inLaemmli sample buffer. Lane a, uninduced pMJ26. Lane b, induced pMJ26.Lanes c-f, purification of ECGF from induced pMJ26. Lane c, supernatant,after removal of cell debris; Lane d, material unabsorbed toheparin-SEPHAROSE in 250 mM NaCl; lane e, entire cell debris pellet oflane c; Lane f, molecular weight standards. Samples in lanes a-dcontained 100 μg protein.

FIGS. 12A, 12B, and 12C illustrate a comparison of human recombinant andbovine brain-derived α-ECGF. --∘--∘--∘-- bovine α-ECGF; --------recombinant human α-ECGF; --□--□--□-- reduced and alkylated recombinanthuman α-ECGF; ▪ recombinant human α-ECGF, no heparin; □ bovine α-ECGF,no heparin.

Panel A. LE-II receptor binding competition assay. Receptor competitionassays were performed. Confluent cultures of LE-II cells were incubatedfor 1.5 h at 4° C. in the present of approximately 5 ng/ml of ²⁵I-bovine α-ECGF and the indicated amounts of unlabelled HPLC-purifiedα-ECGF. Protein concentrations were determined by amino acid analysis.Monolayers were washed three times with DMEM containing 1 mg/ml BSA,lysed with 0.1N NaOH, and the cell-associated radioactivity determined.Binding observed in the absence of competitor is defined as 100%control. Reduced and alkylated recombinant α-ECFG was prepared asfollows: HPLC-purified ECGF in Tris-HCl pH 8.3, 6M guanidinehydrochloride, 100 mM DTT was incubated for 60 minutes at 37° C. undernitrogen. Iodacetic acid was added to 22 mM, and incubation continued inthe dark for 60 minutes at 37° C. The protein was isolated byreversed-phase HPLC. Amino acid composition analysis indicated thepresence of 2.9 mol s-carboxymethyl cysteine/mol α-ECGF.

Panel B. Stimulation of [3H]-thymidine incorporation in LE-II cells.Confluent, murine LE-II cells in DMEM containing 0.1% fetal bovine serumwere incubated with the indicated quantities of bovine or recombinanthuman α-ECGF for 18 hours. Cells were labelled for 4 hours in thepresence of 2.4 μCi [³ H]-thymidine. Wells containing 20% fetal calfserum (X) and 1 mg/ml bovine serum albumin (BSA) served as controls.

Panel C. Human umbilical vein endothelial cell (HUVEC) growth assay.Costar 24 well tissue culture dishes (2 cm² /well) were precoated withhuman fibronectin (10 ug/cm²) in PBS for 0.5-2 hours prior to seedingwith 2×10³ HUVEC in Medium 199 containing 10% fetal bovine serum. Cellswere allowed to attach for 2-4 hours at 37° C., at which time the mediawas aspirated and replaced with 0.75 ml Medium 199 containing 10% fetalbovine serum and, unless otherwise indicated, 5 U/ml heparin. Dilutionsof HPLC-purified recombinant human α-ECGF and bovine brain-derivedα-ECGF in 1-50 μl were added to duplicate wells as indicated. Media werechanged on days 2 and 4, and on day 7 cells were harvested bytrypsinization and cell number was determined with a Coulter counter.Wells containing 20% fetal calf serum (X) and 1 ng/ml BSA served ascontrols.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Introduction

As used herein, recombinant "ECGF" denotes endothelial cell growthfactor or its fragments produced by cell or cell-free culture systems,in bioactive forms having the capacity to influence cellular growth,differentiation, and migration in vitro as does ECGF native to the humanangiogenic process.

Different alleles of ECGF may exist in nature. These variations may becharacterized by differences in the nucleotide sequence of thestructural gene coding for proteins of identical biological function. Itis possible to produce analogs having single or multiple amino acidsubstitutions, deletions, additions, or replacements. All such allelicvariations, modifications, and analogs resulting in derivatives of ECGFwhich retain the biologically active properties of native ECGF areincluded within the scope of this invention.

"Expression vectors" refer to vectors which are capable of transcribingand translating DNA sequences contained therein, where such sequencesare linked to other regulatory sequences capable of affecting theirexpression. These expression vectors must be replicable in the hostorganisms or systems either as episomes, bacteriophage, or as anintegral part of the chromosomal DNA. One form of expression vectorwhich is particularly suitable for use in the invention is thebacteriophage, viruses which normally inhabit and replicate in bacteria.Particularly desirable phage for this purpose are the lambda gt₁₀ andgt₁₁ phage described by Young and Davis, supra. Lambda gt₁₁ is a generalrecombinant DNA expression vector capable of producing polypeptidesspecified by the inserted DNA.

To minimize degradation, upon induction with a synthetic analogue oflactose (IPTG), foreign proteins or portions thereof are synthesizedfused to the prokaryotic protein β-galactosidase. The use of host cellsdefective in protein degradation pathways may also increase the lifetimeof novel proteins produced from the induced lambda gt₁₁ clones. Properexpression of foreign DNA in lambda gt₁₁ clones will depend upon theproper orientation and reading frame of the inserted DNA with respect tothe β-galactosidase promoter and translation initiating codon.

Another form of expression vector useful in recombinant DNA techniquesis the plasmid--a circular unintegrated (extra-chromosomal),double-stranded DNA. Any other form of expression vector which serves anequivalent function is suitable for use in the process of thisinvention.

Recombinant vectors and methodology disclosed herein are suitable foruse in host cells covering a wide range of prokaryotic and eukaryoticorganisms. Prokaryotic cells are preferred for the cloning of DNAsequences and in the construction of vectors. For example, E. coli K12strain HB101 (ATCC No. 33694), is particularly useful. Of course, othermicrobial strains may be used. Vectors containing replication andcontrol sequences which are derived from species compatible with thehost cell or system are used in connection with these hosts. The vectorordinarily carries an origin of replication, as well as characteristicscapable of providing phenotypic selection in transformed cells. Forexample, E. coli can be transformed using the vector pBR322, whichcontains genes for ampicillin and tetracycline resistance [Bolivar, etal., Gene, 2:95 (1977)].

These antibiotic resistance genes provide a means of identifyingtransformed cells. The expression vector may also contain controlelements which can be used for the expression of the gene of interest.Common prokaryotic control elements used for expression of foreign DNAsequences in E. coli include the promoters and regulatory sequencesderived from the β-galactosidase and tryptophan (trp) operons of E.coli, as well as the pR and pL promoters of bacteriophage lambda.Combinations of these elements have also been used (e.g., TAC, which isa fusion of the trp promoter with the lactose operator). Other promotershave also been discovered and utilized, and details concerning theirnucleotide sequences have been published enabling a skilled worker tocombine and exploit them functionally.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,may also be used. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. Yeast promoters suitablefor the expression of foreign DNA sequences in yeast include thepromoters for 3-phosphoglycerate kinase or other glycolytic enzymes.Suitable expression vectors may contain termination signals whichprovide for the polyadenylation and termination of the mRNA transcriptof the cloned gene. Any vector containing a yeast-compatible promoter,origin of replication, and appropriate termination sequence is suitablefor expression of ECGF.

Cell lines derived from multicellular organisms may also be used ashosts. In principle, any such cell culture is workable, whether from avertebrate or invertebrate source. However, interest has been greatestin vertebrate cells, and propagation of vertebrate cells in culture(tissue culture) has become a routine procedure in recent years.Examples of such useful hosts are the VERO, HeLa, mouse C127, Chinesehamster ovary (CHO), WI38, BHK, COS-7, and MDCK cell lines. Expressionvectors for such cells ordinarily include an origin of replication, apromoter located in front of the gene-to be expressed, RNA splice sites(if necessary), and transcriptional termination sequences.

For use in mammalian cells, the control functions (promoters andenhancers) on the expression vectors are often provided by viralmaterial. For example, commonly used promoters are derived from polyoma,Adenovirus 2, and most frequently, Simian Virus 40 (SV40). Eukaryoticpromoters, such as the promoter of the murine metallothionein gene[Paulakis and Hamer, Proc. Natl. Acad. Sci. USA, 80:397-401 (1983)], mayalso be used. Further, it is also possible, and often desirable, toutilize the promoter or control sequences which are naturally associatedwith the desired gene sequence, provided such control sequences arecompatible with the host system. To increase the rate of transcription,eukaryotic enhancer sequences can also be added to the construction.These sequences can be obtained from a variety of animal cells oroncogenic retroviruses such as the mouse sarcoma virus.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as that provided by SV40 orother viral sources, or may be provided by the host cell chromosomalreplication mechanism. If the vector is integrated into the host cellchromosome, the latter is often sufficient.

Host cells can prepare ECGF which can be of a variety of chemicalcompositions. The protein is produced having methionine as its firstamino acid. This methionine is present by virtue of the ATG start codonnaturally existing at the origin of the structural gene or by beingengineered before a segment of the structural gene. The protein may alsobe intracellularly or extracellularly cleaved, giving rise to the aminoacid which is found naturally at the amino terminus of the protein. Theprotein may be produced together with either its own or a heterologoussignal peptide, the signal polypeptide being specifically cleavable inan intra- or extracellular environment. Finally, ECGF may be produced bydirect expression in mature form without the necessity of cleaving awayany extraneous polypeptide.

Recombinant host cells refer to cells which have been transformed withvectors constructed using recombinant DNA techniques. As defined herein,ECGF is produced as a consequence of this transformation. ECGF or itsfragments produced by such cells are referred to as "recombinant ECGF".

B. Recombinant and Screening Methodology

The procedures below are but some of a wide variety of well establishedprocedures to produce specific reagents useful in the process of thisinvention. The general procedure for obtaining an mRNA mixture is toobtain a tissue sample or to culture cells producing the desiredprotein, and to extract the RNA by a process such as that disclosed byChirgwin, et al., Biochemistry, 18:5294 (1979). The mRNA is enriched bypoly(A)mRNA-containing material by chromatography on oligo (dT)cellulose or poly(U) SEPHAROSE, followed by elution of the poly(A)containing mRNA fraction.

The above fraction enriched for poly(A) containing mRNA is used tosynthesize a single-strand complementary cDNA (ss-cDNA) using reversetranscriptase. As a consequence of DNA synthesis, a hairpin loop isformed at the 3' end of the DNA which will initiate second-strand DNAsynthesis. Under appropriate conditions, this hairpin loop is used toeffect synthesis of the ds-cDNA in the presence of DNA polymerase anddeoxyribonucleotide triphosphates.

The resultant ds-cDNA is inserted into the expression vector by any oneof many known techniques. In general, methods can be found in Maniatis,et al., supra, and Methods In Enzymology, Volumes 65 and 68 (1980); and100 and 101 (1983). In general, the vector is linearized by at least onerestriction endonuclease, which will produce at least two blunt orcohesive ends. The ds-cDNA is ligated with or joined into the vectorinsertion site.

If prokaryotic cells or other cells which contain substantial cell wallmaterial are employed, the most common method of transformation with theexpression vector is calcium chloride pretreatment as described byCohen, R. N., et al., Proc. Nat'l. Acad. Sci. USA, 69:2110 (1972). Ifcells without cell wall barriers are used as host cells, transfection iscarried out by the calcium phosphate precipitation method described byGraham and Van der Eb, Virology, 52:456 (1973). Other methods forintroducing DNA into cells such as nuclear injection, viral infection orprotoplast fusion may be successfully used. The cells are then culturedon selective media, and proteins for which the expression vector encodesare produced.

Clones containing part or the entire cDNA for ECGF are identified withspecific oligonucleotide probes deduced from a partial amino acidsequence determination of ECGF. This method of identification requiresthat the non-degenerate oligonucleotide probe be designed such that itspecifically hybridizes to ECGF ds-cDNA. Clones containing ECGF cDNAsequences are isolated by radioactively labeling the oligonucleotideprobe with 32_(p) -ATP, hybridizing the radioactive oligonucleotideprobe to the DNA of individual clones of a cDNA library containingECGF-cDNA, and detection and isolation of the clones which hybridize byautoradiography. Such a cloning system is applicable to the lambda gt₁₁system described by Young and Davis, supra.

Clones containing the entire sequence of ECGF are identified using asprobe the cDNA insert of the ECGF recombinants isolated during theinitial screening of the recombinant lambda gt₁₁ cDNA library withECGF-specific oligonucleotides. Nucleotide sequencing techniques areused to determine the sequence of amino acids encoded by the cDNAfragments. This information may be used to determine the identity of theputative ECGF cDNA clones by comparison to the known amino acid sequenceof the amino-terminus of bovine ECGF and of a peptide derived bycyanogen bromide cleavage of ECGF.

EXAMPLE

A. Preparation of Total RNA

Total RNA (messenger, ribosomal and transfer) was extracted from freshtwo-day old human brain stem essentially as described by Chirgwin,supra, (1979). Cell pellets were homogenized in 5 volumes of a solutioncontaining 4 M guanidine thiocyanate, and 25 mM Antifoam A (SigmaChemical Co., St. Louis, Mo.). The homogenate was centrifuged at 6,000rpm in a SORVALL GSA rotor for 15 minutes at 10° C. The supernatantfluid was adjusted to pH 5.0 by addition of acetic acid and the RNAprecipitated by 0.75 volumes of ethanol at -20° C. for two hours. RNAwas collected by centrifugation and dissolved in 7.5M guanidinehydrochloride containing 2 mM sodium citrate and 5 mM dithiothreitol.Following two additional precipitations using 0.5 volumes of ethanol,the residual guanidine hydrochloride was extracted from the precipitatewith absolute ethanol. RNA was dissolved in sterile water, insolublematerial removed by centrifugation, and the pellets were re-extractedwith water. The RNA was adjusted to 0.2M potassium acetate andprecipitated by addition of 2.5 volumes of ethanol at -20° C. overnight.

B. Preparation of Poly(A)-containing RNA

The total RNA precipitate, prepared as described above, was disolved in20 mM Hepes buffer (pH 7.2) containing 10 mM EDTA and 1% SDS, heated at65° C. for 10 minutes, then quickly cooled to 25° C. The RNA solutionwas then diluted with an equal volume of water, and NaCl was added tobring the final concentration to 300 mM NaCl. Samples containing up to240 A₂₆₀ units of RNA were chromotagraphed on poly(U)-SEPHAROSE usingstandard procedures. Poly(A)-containing RNA was eluted with 70%formamide containing 1 mM Hepes buffer (pH 7.2), and 2 mM EDTA. Theeluate was adjusted to 0.24 M NaCl and the RNA was precipitated by 2.5volumes of ethanol at -20° C.

C. Construction of cDNA Clones in Lambda gt₁₁

The procedure followed for the enzymatic reaction is shown in FIG. 1.The mRNA (20 μg) was copied into ds-cDNA with reverse transcriptase andDNA polymerase I exactly as described by Buell, et al., supra, andWilkensen, et al., J. Biol. Chem., 253:2483 (1978). The ds-cDNA wasdesalted on SEPHADEX G-50 and the void-volume fractions further purifiedon an ELUTIP-D column (Schleicher & Schuell, Keene, N.H.) following themanufacturer's directions. The ds-cDNA was made blunt-ended byincubation with S1 nuclease [Ricca, et al., J. Biol. Chem., 256:10362(1981)]. The reaction mixture consisted of 0.2M sodium acetate (pH 4.5),0.4M sodium chloride, 2.5 mM zinc acetate and 0.1 unit of S1 nucleaseper ng of ds-cDNA, made to a final reaction volume of 100 μl. Theds-cDNA was incubated at 37° C. for one hour, extracted withphenol:chloroform, and then desalted on a SEPHADEX G-50 column asdescribed above.

The ds-cDNA was then treated with EcoRI methylase and Klenow fragment ofDNA polymerase I using reaction conditions described in Maniatis, etal., Molecular Cloning, supra. The cDNA was again desalted on SEPHADEXG-50 as described above and then ligated to 0.5 μg of phosphorylatedEcoRI linkers using T4 DNA ligase (Maniatis, et al., supra). The mixturewas cleaved with EcoRI and fractionated on an 8% acrylamide gel inTris-borate buffer (Maniatis, et al., supra). DNA with a size greaterthan 1 kilobase was eluted from the gel and recovered by binding to anELUTIP-D column, eluted with 1M NaCl and then collected by ethanolprecipitation.

As shown in FIG. 2, the DNA fragments were then inserted into EcoRIcleaved and phosphatase-treated lambda gt₁₁, using T4 DNA ligase. Alibrary of 5.7×10⁶ phage was produced, of which approximately 65% wererecombinant phage. The library was amplified by producing plate stocksat 42° C. on E. coli Y1088 [supE supF metB trpR hsdR⁻ hsdM+tonA21 strAlacU169 (proC::Tn5) (pMC9)]. Amplification procedures are described inManiatis, et al., supra. Important features of this strain, described byYoung and Davis, supra, include (1) supF (required suppression of thephage amber mutation in the S gene), (2) hsdR- hsdM+ (necessary toprevent restriction of foreign DNA prior to host modification), and (3)lacU169 (proC::Tn5), and (4) (pMC9) (a Lac I-bearing pBR322 derivativewhich represses, in the absence of an inducer, the expression of foreigngenes that may be detrimental to phage and/or cell growth).

D. Identification of Clones Containing ECGF Sequence

To screen the library for recombinant phage containing ECGF cDNA,1.5×10⁶ phage were plated on a lawn of E. coli Y1090 [delta lacU169proAIon araD139 strA supF (trpC22::TnlO) (pMC9)]and incubated at 42° C. for6 hours. After the plates were refrigerated overnight, a nitrocellulosefilter was overlaid on the plates. The position of the filter was markedwith a needle. The filter removed after one minute and left to dry atroom temperature. From each plate, a duplicate filter was preparedexactly as described, except that the filter was left in contact withthe plate for 5 minutes. All filters were then prepared forhybridization, as described in Maniatis, et al., supra. This involvedDNA denaturation in 0.5M NaOH, 1.5M NaCl, neutralization in 1M Tris-HCl,pH 7.5, 1.5M NaC1, and heating of the filters for 2 hours at 80° C. invacuo.

To screen the human brain stem cDNA library for clones containing ECGFinserts, a specific oligonucleotide was designed. This oligonucleotidewas based upon a partial amino acid sequence analysis of the aminoterminus of ECGF. As shown in FIG. 3, lines a & b, bovine ECGF isisolated as two species, designated alpha and beta ECGF, which differonly in the amino acids found at the respective amino termini. As shownin FIG. 3, line b, beta-ECGF is a slightly larger species thanalpha-ECGF. The exact amino acid sequence at the amino terminus ofbeta-ECGF is undetermined, however, a sequence derived from fast atombombardment mass spectral analysis and the amino acid composition of theamino terminal tryptic peptide of bovine beta-ECGF is shown. The aminoterminal blocking group appears to be acetyl. If intact beta-ECGF iscleaved by trypsin, a second amino amino acid sequence found in beta butnot alpha ACGF starting with PheAsnLeu . . . is determined. Thissequence is also found at the amino terminus of acidic fibroblast growthfactor [Thomas, K. A. et al., Prac. Natl. Acad. Sci. USA, 82:6409-6413(1985)]. The amino terminus of alpha-ECGF is AsnTyrLys . . . (FIG. 3,line a) and is the equivalent of beta-ECGF minus an amino terminalextension. In FIG. 3, lines c and d set forth for comparison the aminoacid sequence of cyanogen bromide-cleaved bovine alpha and beta ECGF,respectively.

For oligonucleotide design, the amino acid sequenceIleLeuProAspGlyThrValAspGlyThrLys, corresponding to alpha-ECGF aminoacids 19-29 inclusive, was chosen. Rather than design a mixture ofoligonucleotides covering all of the possible coding sequences (owing tothe degeneracy of the genetic code), a long unique oligonucleotide wasdesigned. Such oligonucleotide probes have been previously shown to besuccessful probes in screening complex cDNA [Jaye, et al., Nucleic AcidsResearch 11:2325-2335, (1983)] and genomic [Gitschier, et al., Nature,312:326-330 (1984)] libraries. Three criteria were used in designing theECGF probe: (1) The dinucleotide CG was avoided. This strategy was basedupon the observed under representation of the CG dinucleotide ineukaryotic DNA [Josse, et al., J. Biol. Chem. 236:864-875, (1961)]; (2)preferred codon utilization data was used wherever possible. A recentand comprehensive analysis of human codon utilization was found inLathe, J. Mol. Biol. 183:1-12 (1985); and (3) wherever the strategies ofCG dinucleotide and preferred codon utilization were uninformative,unusual base pairing was allowed. This strategy was based upon thenatural occurence of G:T, I:T, I:A and I:C base pairs which occur in theinteraction between tRNA anticodons and mRNA codons [Crick, J. Mol.Biol. 19:548-555, (1966)]. A diagram of usual and unusual base pairs isshown in FIG. 4. Use of I (Inosine) in a hybridization probe was firstdemonstrated, in a model experiment, by Ohtsuka, et al., J. Biol. Chem.260:2605-2608 (1985). The overall strategy and choice made in the designof the oligonucleotide used to screen the human brain stem cDNA libraryfor ECGF is shown in FIG. 5. In addition, two other oligonucleotides,designed with the same strategy, were constructed.

Approximately 30 pmole of the oligonucleotide shown in FIG. 5 wereradioactively labeled by incubation with ³² P-γ-ATP and T4polynucleotide kinase, essentially as described by Maniatis, et al.,supra. Nitrocellulose filters, prepared as described above, wereprehybridized at 42° C. in 6× SSPE (1× SSPE =0.18M NaCl, 0.001M NaHPO₄pH 7.2, 0.001M EDTA), 2× Denhardt's (1× Denhardt's--0.02% each FICOLL,polyvinylpyrrolidone, bovine serum albumin), 5% dextran sulfate, and 100mu g/ml denatured salmon sperm DNA. The ³² P-labeled oligonucleotide wasadded following four hours of prehybridization, and hybridizationcontinued overnight at 42° C. Unhybridized probe was removed bysequential washing at 37° C. in 2× SSPE, 0.1% SDS.

From 1.5×10⁶ plaques screened, 2 plaques gave positive autoradiographicsignals after overnight exposure. These clones were purified tohomogeneity by repeated cycles of purification using the aboveoligonucleotide as hybridization probe.

The two clones that were isolated, ECGF clones 1 and 29, were analyzedin further detail. Upon digestion with EcoRI, clone 1 and 29 revealedcDNA inserts of 2.2 and 0.3 Kb, respectively. Nick translation of clonedcDNA and its subsequent use as a radiolabeled probe in Southern blotanalysis (Maniatis, et al., supra) revealed that clones 1 and 29 wererelated and overlapping clones. The overlapping nature of these twoclones is shown in FIG. 6.

Clones 1 and 29 were analyzed in further detail as follows: Anadditional two oligonucleotides were designed, based upon the amino acidsequence of bovine ECGF. These oligonucleotides were designed based uponthe same considerations as those used in the design of theoligonucleotide used to isolate clones 1 and 29. These oligonucleotides(ECGF oligonucleotides II and III) are shown in FIG. 7. These twooligonucleotides as well as oligo(dT)18 were radioactively labeled in akination reaction as described above and used as hybridization probes inSouthern blotting experiments. The results of these experiments showedthat the 0.3 Kb cDNA insert of clone 29 hybridized to ECGFoligonucleotides I and II but not to ECGF oligonucleotide III oroligo(dT)18; the 2.2 Kb cDNA insert of clone 1 hybridized tooligonucleotide I, II, III as well as oligo(dT)18. These data andsubsequent nucleotide sequence determination of clones 1 and 29 showedthat the 3' end of clone 1 ends with a poly(A) tail. Hybridization ofclone 1 to ECGF oligonucleotide III, which is based on a cyanogenbromide cleavage product of bovine ECGF, as well as to oligo(dT)18,strongly suggested that this clone contains the rest of the codingsequence for both alpha and beta ECGFs as well as a large (greater than1 Kb) 3' flanking sequence.

The cDNA inserts from clones 1 and 29 were isolated, subcloned intoM13mp18, and the ECGF-encoding open reading frame and flanking regionssequenced by the chain termination method [Sanger et al., Proc. Natl.Acad. Sci. USA 74:5463-5467 (1977)]. The nucleotide sequence of theseclones and the amino acid sequence deduced from the nucleic acidsequence is shown in FIG. 8. Examination of the nucleotide sequencereveals an open reading frame of 465 nucleotides encoding human ECGF.The 155 amino acids of human ECGF were found to be flanked bytranslation stop codons. The NH₂ -terminal amino acid of human beta ECGFdeduced from the cDNA sequence is methionine, which most likely servesas the translation initiation residue. These data, together with therelatively non-hydrophobic nature of the first 15-20 amino terminalresidues, strongly suggest that human beta ECGF is synthesized without aNH₂ -terminal signal peptide. A comparison of FIGS. 3 and 8 shows thatthe amino terminal amino acid sequence of trypsin-cleaved bovine betaECGF as well as that of bovine alpha ECGF are nearly identical to theamino acid sequence predicted from the nucleotide sequence of lambdaECGF clones 1 and 29. An overall homology between the two species ofover 95% is observed.

Northern blot analysis (Maniatis, et al, supra) reveals that ECGF mRNAis a single molecular species which comigrates with 28S rRNA (FIG. 9).Considering the variation in the estimated size of 28S rRNA, theapproximate size of ECGF mRNA is 4.8±1.4 Kb. All of the sequenceencoding the mature forms of both alpha and beta ECGF is encoded withinECGF clones 1 and 29, which together encompasses approximately 2.3 Kb.Thus, these data demonstrate that the region 5' and flanking theECGF-encoding sequences, is very large (approximately 2.5±1.4 Kb).

cDNA inserts from clone 1 and clone 29 were excised by digestion withEcoRI and subcloned in pUC8 at the EcoRI site. The plasmid formed fromclone 1 was designated pDH15 and the plasmid formed from clone 29 wasdesignated pDH14.

Clone 1 was improved by inserting it into a vector allowing moreefficient expression of α-ECGF. This vector is pMJ26 and places thisgene under a high-effeciency tac promoter as described in FIG. 10 and asdone as follows. A double-stranded Bam HI cohesive 66-meroligonucleotide encoding residues 1-19 of α-ECGF, preceded by initiatormethionine, was synthesized by the phosphoramoridite method andpurified. The oligonucleotide was ligated between the Bam HI sites ofpDH15 creating pMJ25. In order to introduce appropriate regulatorysequences, the α-ECGF-encoding open reading frame was excised from pMJ25by digestion with Eco RI and Hinc II and cloned between the Eco RI andSmaI sites of pKK223-3 (PL Biochemicals). The recombinant plasmid,pMJ26, was introduced into the Laci q bearing E. coli strain, JMTO3, toevaluate expression of α-ECGF.

In pMJ26, expression of α-ECGF, under control of the hybrid tacpromoter, is inducible with IPTG. To measure α-ECGF production,logarithmically grown bacterial cultures containing pMJ26 at A₅₅₀ of 0.2were induced with 1 mM IPTG and grown for 2-4 hours at 37° C. prior toharvesting, lysis and growth factor isolation. Control extracts wereprepared from uninduced cultures of pMJ26 and from induced and uninducedbacterial cultures not containing the ECGF gene. All extracts werefractionated by SDS-PAGE, and the protein visualized by staining withCoomassie brilliant blue. As shown in FIG. 11, lane b, a prominant bandat approximately 16 Kd is observed in induced cultrues of pMJ26. Theband is observed at low levels when pMJ26 is not induced, lane a, (thisreflects the leakiness of the tac promoter) and, as expected, is absentin either induced or control cultures of bacterial which do not containthe α-ECGF gene.

The ability to induce a polypeptide of the expected size, specifically,in bacteria containing the α-ECGF gene, suggests the successfulexpression of the human α-ECGF. The protein was purified by a two-stepprocedure involving heparin-SEPHAROSE column chromatography follwed byreversed phase HPLC analysis. (Burgess, W. H., Mehlman, T., Friesel, R.,Johnson, W. V., and Maciag, T. (1985) J. Biol. Chem. 260, 11389-11392.)Protein evaluated by this method is essentially pure and amino terminaland amino acid sequence analyses demonstrate the predicted amino acidsequence of α-ECGF of MNYKKPKLLYCSNG. Data suggest (FIG. 11) pMJ26 canexpress α-ECGF to approximately 10% of the total protein of E. coli andremain soluble in this bacteria allowing this rapid two-steppurification. To establish that this protein is biologically active, itwas compared to bovine ECGF in several established assays.

In these assays, the functional activities of recombinant human α-ECGFwere examined. The success of the heparin-SEPHAROSE affinity basedpurification demonstrates that recombinant α-ECGF binds to immobilizedheparin. In addition, heparin potentiates the mitogenic activity ofrcombinant α-ECGF (FIG. 12B). Together these data indicate that theheparin binding properties of the recombinant material are similar tothose of bovine brain-derived ECGF.

The results of cellular receptor assays (Friesel, R., Burgess, W. H.,Mehlman, T., and Maclag, T. (1986) J. Biol. Chem. 261, 7581-7584;Schreiber, A. B., Kenney, J., Kawalski, J., Firesel, R., Mehlman, T.,and Maciag, T. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6138-6143)indicate that the receptor binding activity of recombinant human α-ECGFalso is similar to bovine brain-derived ECGF. Radioiodinated bovineα-ECGF was incubated with murine endothelial cells at 4° C. in thepresence of increasing quantities of either bovine or recombinant humanα-ECGF. After 30 minutes, the cell monolayer was washed and thecell-associated radioactivity determined. As shown in FIG. 12a, thedisplacement curves for both bovine and human recombinant α-ECGF arevery similar. The receptor-binding activity of the recombinant proteinwas abolished after reduction and alkylation (FIG. 12a).

The mitogenic activities of native and recombinant α-ECGF were in twoseparate assays. In the first assay DNA synthesis was monitored byincorporation of [³ H]-thymidine into TCA-precipitable material as afunction of increasing quantities of α-ECGF (FIG. 12b). The second assaycompared the stimulation of both preparations of ECGF upon theproliferation of HUVEC (FIG. 12C). In the [³ H]-thymidine incorporationassay (FIG. 12B), the maximal response observed with the recombinantmaterial was identical to that observed with bovine brain-derived ECGF,while the dose for each which gave half-maximal stimulation was similar(EC₅₀ of bovine α-ECGF=1.75 ng/ml; EC₅₀ of recombinant human α-ECGF=0.5ng/ml). In the HUVEC assay (FIG. 12C), the maximal stimulation observedwith bovine and recombinant human ECGF were similar, as were theconcentrations giving half-maximal stimulation (EC₅₀ of bovineα-ECGF=0.6 ng/ml; EC₅₀ of recombinant human α-ECGF=0.45 ng/ml). Heparin(5 U/ml) was found to potentiate the mitogenic effect of both bovine andrecombinant human α-ECGF 5-10 fold. These date demonstrate that humanrecombinant α-ECGF has biological properties similar to bovine ECGF.

Thus, this example describes experimental procedures which provide humanendothelial cell growth factor essentially free of other proteins ofhuman origin.

ECGF has utility in the growth and amplification of endothelial cells inculture. Currently, ECGF for cell culture use is extracted-from bovinebrain by the protocol of Maclag, et al., [Proc. Natl. Acad. Sci. U.S.A.,76:11, 5674-5678 (1978)]. This crude bovine ECGF is mitogenic for humanumbilical vein endothelial cells [Maciag, et al., J. Biol. Chem.257:5333-5336 (1982)]and endothelial cells from other species.Utilization of heparin with ECGF and a fibronectin matrix permits theestablishment of stable endothelial cell clones. The recommendedconcentration of this crude bovine ECGF for use as a mitogen in vitro is150 micrograms per milliliter of growth medium.

Recombinant DNA-derived human ECGF has utility, therefore, as animproved substitute for crude bovine ECGF in the in vitro culturing ofhuman endothelial cells and other mesenchymal cells for research use.The activity of human ECGF is expected to be the same as or better thanbovine ECGF in the potentiation of endothelial cell growth due to thehigh degree of homology in the amino acid sequences of both proteins.The expected effective dose range for potentiating cell division andgrowth in vitro is 5-10 ng of purified ECGF per milliliter of culturemedium. Production of the ECGF via recombinant-DNA technologies asoutlined in this patent application and subsequent purification asdescribed by Burgess, et al., [J. Biol. Chem. 260:11389-11392 (1985)]will provide large quantities of a pure product of human origin(heretofore unavailable in any quantity or purity) with which to developmodels of human homeostatis and angiogenesis.

Recombinant DNA-derived human ECGF also has utility in the potentiationof cell growth on a prosthetic device, rather than a tissue cultureflask or bottle. This device may or may not be coated with othermolecules which would facilitate the attachment of endothelial cells tothe device. These facilitating molecules may include extracellularmatrix proteins (e.g. fibronectin, laminin, or one of the collagens),human serum albumin, heparin or other glycosaminoclycans or inertorganic molecules. Endothelial cells would be cultured on these surfacesusing effective doses of ECGF in the culture medium, ultimately coveringthe device with an endothelial cell monolayer. This device would thenprovide a non-thrombogenic surface on the prosthetic device, thusreducing the risk of potentially life-threatening thrombogenic eventssubsequent to implantation of the prosthetic device.

ECGF has utility in diagnostic applications. Schreiber, et al., [Proc.Natl. Acad. Sci. USA 82:6138 (1985)] developed a double antibodyimmunoassay for bovine ECGF. In this assay, 96-well polyvinyl chlorideplates were coated with rabbit anti-ECGF and the remaining binding sitessubsequently blocked with 10% normal rabbit serum. Samples of ECGF werethen added to the wells and incubated. After washing, murine monoclonalanti-ECGF was added. After incubation and several washes, rabbitanti-mouse IgG coupled with peroxidase was added. The reaction productwas quantitated spectrophotometrically after conversion ofO-phenylenediamine in the presence of hydrogen peroxide. A similarlyconstructed immunoassay may be useful for monitoring human ECGF levelsin disease states affecting endothelial cell growth. Purifiedrecombinant-DNA derived ECGF would be useful as a standard reagent inquantifying unknown ECGF samples.

ECGF also may have potential in the treatment of damaged or in theregeneration of blood vessels and other endothelial cell-linedstructures.

It should be appreciated that the present invention is not to beconstrued as being limited by the illustrative embodiment. It ispossible to produce still other embodiments without departing from theinventive concepts herein disclosed. Such embodiments are within theability of those skilled in the art.

Deposit of Strains Useful in Practicing the Invention

Biologically pure cultures of strains for practicing this invention areavailable at the offices of Rorer Biotechnology Inc.

Access to said cultures will be available during pendency of the patentapplication to one determined by the Commissioner to be entitled theretounder 37 C.F.R. Section 1.14 and 35 U.S.C. Section 122.

At a date prior to issuance a deposit of biologically pure cultures ofthe strains within the allowed claims will be made with the AmericanType Culture Collection, 12301 Parklawn Drive, Rockville, Md., theaccession number assigned after successful viability testing will beindicated by amendment below, and the requisite fees will be paid.

All restriction on availability of said culture to the public will beirrevocably removed upon the granting of a patent based upon theapplication and said culture will remain permanently available for aterm of at least five years after the most recent request for thefurnishing of a sample and in any case for a period of at least 30 yearsafter the date of the deposit. Should the culture become nonviable or beinadvertently destroyed, it will be replaced with a viable culture(s) ofthe same taxonomic description.

    ______________________________________                                        Strain/Plasmid                                                                              ATCC No.  Deposit Date                                          ______________________________________                                        pDH 15        53336     November 25, 1985                                     pDH 14        53335     November 25, 1985                                     pMJ 26        67857     November 23, 1985                                     ______________________________________                                    

What is claimed is:
 1. Isolated and purified biologically active bovineβ endothelial cell growth factor having an amino terminal sequence ofAla Glu Gly Glu Thr Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe Asn Leu ProLeu Gly, an apparent molecular weight of 20,000 and having a specificactivity for promoting human endothelial cell growth of at least theactivity obtained when purified 16,300 fold from bovine brain extract.